There presently exists about 75,000 synthetic chemicals in commercial use, with the number increasing annually by about 1,000. All of these chemicals must be shown to be non-toxic or of limited toxicity before being introduced into the environment. In addition waste materials of advanced societies are accumulating at an exponential rate, all of which must be either deposed in land fill or some equivalent storage site, or treated to reduce their bulk or toxicity, and then released into the environment. As is the case with new synthetic chemicals, the toxic potential of all waste materials, either before or after treatment, must be known to permit responsible and ecologically sound management practices. The need for reliable means for testing toxicity is therefore great, and this need has been recognized for many years.
The procedure for genotoxicity testing is well established, and involves, generally speaking, a series of tests at increasing levels of complexity and expense: in vitro mutational assays on bacteria, mutation and/or transformation assays on cultured mammalian cells and then a variety of in vivo tests in animals, both short and long term. The choice of the battery of in vitro tests is important, as false negative results at this stage result in substantial expenses in time and money in undertaking the more elaborate animal studies. In these in vitro tests, bacterial or mammalian cells which carry a single functional copy of a given gene in their genetic make-up are treated with a suspected mutagen that some of the cells in the population may become mutant in this gene. This mutation changes the sequence of base pairs in the gene or biochemical deficiency within the cell. This cell is then selected for by growth in a medium containing an agent lethal for wild type cells, but innocuous to cells with the said deficiency. The number of mutant cell colonies obtained per million cells plated in the selective medium reflects the mutagenic activity. These producers are presented in detail in U.S. Pat. Nos. 4,066,510 and 4,302,535 issued to W. G. Thilly and T. R. Skepek et al, respectively.
One shortcoming of many of the cell strains in use is that the genetic locus carrying the gene to be assayed is hemizygous; that is the genetic sequences surrounding the aforesaid gene are present in only one copy per cell. For example, in the CHO/HGPRT assay developed specifically for genotoxicity testing, the hypoxanthine guanine phosphoribosyltransferase (HGPRT) gene is on the X-chromosome, which is present in only one copy in CHO cells. If a genotoxic agent induces large genetic deletions removing many genes including HGPRT (these deletions are referred to as multilocus events), a neighbouring gene whose protein product may be essential to the growth of the cell will also be removed and this will kill the cell.
A limited number of assay systems have been developed to overcome this shortcoming, Two cell lines, the mouse lymphocyte L5178Y line and the human lymphocyte TK6 line are presumed to be heterozygous at the thymidine kinase (TK) locus; that is, one of the two functional copies of the TK gene present in the parental cell has already been made non-functional by a small mutational event affecting the TK gene and no neighbouring genes. Mutant events occurring at the remaining normal locus may remove neighbouring sequences including any essential genes present, but even so a second, functional copy of each of these genes remains associated with the other copy of the TK gene which had previously been mutated. The cells made mutant at the second TK gene can therefore grow even if the mutation is a multilocus event.
An alternative cell line, called AS52, has been developed in which a single copy of a gene derived from bacteria, xanthine guanine phosphoribosyltransferase (gpt) was artificially introduced into the genome of a mammalian cell. The region of insertion is present in two copies in this cell line. These cells can be treated with a suspected mutagen, and any events which inactivate or remove the gpt gene can be detected by growing the treated cells in the appropriate selective medium, and multilocus events will not kill the cells because a second copy of each of the neighbouring genes remains in the cell.
A fourth cell line, D423, has been proposed as having the potential of overcoming the deficiencies of hemizygous cell lines. This is a so-called class III heterozygote, partially deficient in the level of functional adenine phosphoribosyltransferase (APRT). In a fashion similar to the above-mentioned TK.sup.+/- heterozygous lines, this line undergoes mutation to resistance to the drug 2,6 diaminopurine, and agents which induce multilocus deletions can induce mutations at the functional APRT gene without killing the cell. It is not clear how closely mutation in this cell reflects the process of mutation in normal cells, however, since 1) one of the chromosomes carrying APRT has been rearranged in D423 and 2) previous molecular analysis suggested there may be three copies of the APRT gene in this line.
An important part of the protocol in measuring mutation induced by a suspected mutagen is to perform the control experiment. A portion of the same population cells used for mutagen treatment is grown in the selective medium without such a treatment, in order to determine the number of mutants pre-existing in the population. This is called the spontaneous mutant frequency and it depends mostly on the probability that a given cell will undergo spontaneous mutation at the locus in question during a given cell cycle. This in turn is called the spontaneous mutation rate, and can be measured by the Luria-Delbruck fluctuation test. The sensitivity of a given mutagen assay depends directly on the level of the background of spontaneous mutants in the population, since a mutagenic effect cannot be detected unless the number of mutant colonies determined after treatment is higher than this background by a statistically significant amount.
One shortcoming of some of the heterozygous mammalian cell lines in use (including AS52) is that the spontaneous frequencies of mutation are high: the line L5178Y is variously reported to have spontaneous TK mutants at 3.5.times.10.sup.-5 to 4.times.10.sup.-4 /cell; AS52 cultures have gpt mutants at between 3.times.10.sup.-5 and 10.sup.-4 /cell. TK6 cells are reportedly more stable, with spontaneous TK mutant frequencies at between 1 and 4.times.10.sup.-6. This is achieved artificially, by first growing cells in a selective medium which kills mutant TK cells. The D423 line has a spontaneous mutation rate at the APRT locus at 3.times.10.sup.-7, as measured by the Luria-Delbruck assay; two other class III APRT heterozygotes, D424 and D425, also have rates &lt;5.times.10.sup.-7 /cell/generation. Cultures of these cell lines usually have background incidences of mutants of &lt;10.sup.-6. Thus D423, D424 and D425 are particularly well suited to genotoxicity testing in this respect.
A refinement of the above-described procedure to determine genotoxicity of various agents consists of analysis of the nucleotide sequences of mutant cells to determine the nature of the mutational event. For example, to distinguish between point (small) mutations and multilocus events, an analysis of whether one or both copies of the mutated gene are still present in the cell can be made. This is possible if a known polymorphism exists which distinguishes the two sequences coding the for two copies of the gene being analysed. Such a polymorphism was reported near the TK gene in the cell line TK6, but its existence has not been confirmed by molecular analysis.